Brandon:Protocols/Cloning Transformation3: Difference between revisions
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>Bsos (Created page with "==cloning and A-tailing etc.== 1. reaction from the manual transfect into E. coli, use transfection protocol 1. Thaw one vial of One Shot TOP10 chemically competent cel...") |
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==cloning and A-tailing etc.== | ==cloning and A-tailing etc.== | ||
*procedure for TA cloning and tranformation of E. Coli | |||
===Cloning into vector=== | |||
*make sure you know to use TA cloning or blunt cloning vector. | |||
*procedure for cloning with Invitrogen TA cloning kit, for TA cloning. | |||
1. amount of PCR product needed. = ((200)(50))/3900 = 2.5 ng | |||
2. reaction mixture | |||
3 uL PCR product | |||
1 uL ligation buffer | |||
2 uL pCR vector 2.1 (25 ng/ul) | |||
1 uL nuclease free water | |||
3 uL T4 DNA ligase (used 1U/uL T4 not from kit, Kit T4 is @ 5U/uL | |||
10 uL total | |||
3. incubate at 14C for 4 hours or overnight preferably | |||
4. Now ready for transformation into E coli. | |||
===Transformation of E. coli=== | |||
*transfect into E. coli, use transfection protocol | |||
1. Thaw one vial of One Shot TOP10 chemically competent cells for each transformation | 1. Thaw one vial of One Shot TOP10 chemically competent cells for each transformation | ||
Add 1-3 uL of PCR product to TOP10 cell vial (uL based on calculation) | Add 1-3 uL of PCR product/ligated cloning vector from above to TOP10 cell vial (uL based on calculation), | ||
MIX GENTLY, no pipetting up and down | MIX GENTLY, no pipetting up and down | ||
Revision as of 00:08, 16 May 2012
cloning and A-tailing etc.
- procedure for TA cloning and tranformation of E. Coli
Cloning into vector
- make sure you know to use TA cloning or blunt cloning vector.
- procedure for cloning with Invitrogen TA cloning kit, for TA cloning.
1. amount of PCR product needed. = ((200)(50))/3900 = 2.5 ng 2. reaction mixture 3 uL PCR product 1 uL ligation buffer 2 uL pCR vector 2.1 (25 ng/ul) 1 uL nuclease free water 3 uL T4 DNA ligase (used 1U/uL T4 not from kit, Kit T4 is @ 5U/uL 10 uL total 3. incubate at 14C for 4 hours or overnight preferably 4. Now ready for transformation into E coli.
Transformation of E. coli
- transfect into E. coli, use transfection protocol
1. Thaw one vial of One Shot TOP10 chemically competent cells for each transformation Add 1-3 uL of PCR product/ligated cloning vector from above to TOP10 cell vial (uL based on calculation), MIX GENTLY, no pipetting up and down 2. incubate vials on ice for 30 minutes 3. Heat-shock cells for 30 seconds at 42C, no shaking 4. remove from 42C and place on ice for 2 minutes 5. add 250 ul pre-warmed SOC medium to each vial 6. cap vials tightly and shake horizontally at 37C for 1 hr at 224 rpm in shaking incubator 7. spread 20-200 ul of transformations onto pre-warmed selective plates and incubate overnight at 37C. *use 2-3 different concentrations, 1:10, 50 uL, 200 uL or w/e 8. Store reamining transforamtion mix at 4C. cells can be plated next day. 9. Invert selective plates, incubate at 37 overnight. 10. Select colonies and analyze with PCR or w/e.